Gene targeting in mice to study the role of neuropeptide receptors in ethanol dependence has recently yielded fascinating results. Knockout of the mu opioid receptor (MOP) gene blocks ethanol drinking and operant responding for ethanol. Knockout of the Corticotropin Releasing Factor receptor 1 (CRF1) gene reduces levels of anxiety under basal and alcohol withdrawal conditions. Altogether data demonstrate that blockade of these receptor systems reduce alcohol intake. Limitations of these """"""""conventional"""""""" gene targeting studies are that (i) gene knockout occurs early, therefore compensatory mechanisms could take place during development, and (ii) knockout of the receptors occurs throughout the entire animal, therefore no information on the recruited neurocircuitry is provided. To address these issues, we will induce the knockout of MOP and CRF1 receptor genes specifically in the extended amygdala (EA) of adult animals, based on the overall hypothesis of INIA (Integrative Neuroscience Initiative on Alcoholism) regarding the role of the EA in excessive alcohol consumption. First, we will take advantage of two existing mutant mouse lines, one with a floxed MOP receptor gene (recently created in our laboratory), and another with a floxed CRF1 receptor gene (collaboration). Second, we will develop a novel transgenic mouse line expressing Cre recombinase in the EA. To do this, we will use a BAG promoter for the WFS1 (Wolfram syndrom 1) gene, that we have recently identified as an EA marker gene (Specific Aim 1). Third, we will breed floxed mice with the WFS1-Cre mouse to produce the conditional knockout of MOP (Specific Aim 2) and CRF1 (Specific Aim 3) receptor genes in the EA of adult mice. The two conditional lines will be fully characterized for receptor expression throughout the brain, for morphine responses (MOP) and for basal behaviors (Specific Aims 2 and 3). The two conditional lines will finally be extensively studied in behavioral models of excessive alcohol drinking, including the DID and WID models, as well as for acute ethanol responses and ethanol withdrawal (Specific Aim 4). Importantly, the WFS1-Cre transgenic mice generated in Specific Aim 1 will represent a unique tool for the conditional deletion of any other gene of interest in the extended amygdala, and will be generally useful in addiction research.
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